Method of providing  human tumor-specific antibodies

ABSTRACT

Provided are novel tumor-specific binding molecules, particularly human antibodies as well as fragments, derivatives and variants thereof that recognize tumor-associated antigens and that are obtained from a tumor patient who shows at least partial clinical response or is symptom-free. In addition, pharmaceutical compositions comprising such binding molecules, antibodies and mimics thereof and methods of screening for novel binding molecules, which may or may not be antibodies as well as targets in the treatment of tumors are described.

FIELD OF THE INVENTION

The present invention generally relates to novel tumor-specific bindingmolecules of human origin, particularly human antibodies as well asfragments, derivatives and variants thereof that recognize tumorantigens and tumor-associated antigens, respectively. In addition, thepresent invention relates to compositions comprising such bindingmolecules, antibodies and mimics thereof, and to methods of screeningfor novel binding molecules, which may or may not be antibodies, targetsand drugs in the treatment of various tumors, in particular melanoma,breast cancer and metastasis.

BACKGROUND OF THE INVENTION

It is fairly well established that many pathological conditions, such asinfections, cancer, autoimmune disorders, etc., are characterized by theinappropriate expression of certain molecules. These molecules thusserve as “markers” for a particular pathological or abnormal condition.Apart from their use as diagnostic “targets”, i.e., materials to beidentified to diagnose these abnormal conditions, the molecules serve asreagents which can be used to generate diagnostic and/or therapeuticagents. A by no means limiting example of this is the use of cancermarkers such as tumor-associated antigens to produce antibodies specificto a particular marker.

Humoral immune responses to tumors occur in a relatively high frequencyin (1, 2). This phenomenon was exploited to identify a variety oftumor-associated antigens (taa) by screening autologous expressionlibraries with serum from cancer patients (1). Several of these taa nowserve as T cell antigens for the induction of anti-tumor CTL-responsesin patients (3, 4). This preference for the cellular-, in most casescytotoxic immune response as therapeutic strategy is now beingreconsidered and novel vaccines are designed to also induce antibodyresponses. In part, this change of concept may have been influenced bythe recent success of various monoclonal antibodies for tumor therapysuch as trastuzumab (Herceptin) and bevacizumab (Avastin) (5). Whilethese monoclonal antibodies had been specifically raised against targetsof presumed oncological relevance, antibodies occurring in cancerpatients, either spontaneously or upon vaccination form a differentclass of molecules the therapeutic significance of which had beendifficult to assess. This is mostly due to the lack of straightforwardexperimental approaches for their isolation and subsequentcharacterization in vitro and in animal models of human cancer.

Thus, there is a need of providing means and methods to overcome theabove-described limitations.

SUMMARY OF THE INVENTION

Object of the present invention is a method for identifying, validatingand producing tumor-specific diagnostically and therapeutically usefulbinding molecules, in particular antibodies that are directed againstantigens of and/or associated with tumor cells and cancerous tissue, andwhich overcome the drawbacks of murine derived antibodies such as HAMA(human anti-murine antibody) response. More specifically, the presentinvention relates to consistent further development and thusconfirmation of the general method of providing disease-specific bindingmolecules and targets as disclosed in applicant's co-pendinginternational application PCT/EP2008/000053, the disclosure content ofwhich is incorporated herein by reference. Thus, in one aspect thepresent invention relates to a method for isolating a human bindingmolecule specific for a tumor antigen or tumor-associated antigencomprising:

-   (a) subjecting a sample obtained from a patient bearing a tumor    antigen or tumor-associated antigen positive tumor wherein said    patient shows a at least partial clinical response or is    symptom-free to a specimen of tumor cells or tissue of predetermined    clinical characteristics; and-   (b) identifying and optionally isolating an antibody which binds to    said specimen but not to corresponding cells or tissues of a healthy    subject.

The present invention makes use of the tumor-specific immune response ofcancer patients for the isolation of tumor antigen and tumor-associatedantigen (taa) specific human monoclonal antibodies. In particular,experiments performed in accordance with the present invention weresuccessful in the isolation of a monoclonal antibody specific for thetaa NY-ESO-1 from a melanoma patient who showed a serum titer toNY-ESO-1 and a partial clinical response. For isolating the humanantibody specific for a tumor antigen and taa, respectively, the methodof the present invention makes use of immunohistochemistry (IHC) usingtissue microarrays (TMA).

The present invention is thus directed to human antibodies,antigen-binding fragments and similar antigen binding molecules whichare capable of recognizing tumor antigen and tumor-associated antigen(taa), respectively. Furthermore, the present invention relates tocompositions comprising said antibodies and to immunotherapeutic andimmunodiagnostic methods using the same.

Since the present invention enables identifying and isolating moleculartarget structures in cancerous cells and tissues, a further embodimentconcerns novel tumor antigens and tumor associated antigens, which arecharacterized by their property of being bound by the humantumor-specific antibody of the present invention with high affinity.

Naturally, the present invention extends to the immortalized human Bmemory lymphocyte and B cell, respectively, that has been isolated inthe course of performing the method of the present invention andproduces the human antibody.

The present invention also relates to polynucleotides encoding at leasta variable region of an immunoglobulin chain of the antibody of theinvention. Accordingly, the present invention also encompasses vectorscomprising said polynucleotides and host cells transformed therewith aswell as their use for the production of an antibody and equivalentbinding molecules which are specific for antigens that are indicativeand/or causative for a tumor, in particular for melanoma or breastcancer.

The antibody, immunoglobulin chain(s), binding fragments thereof andantigen binding to said antibody can be used in pharmaceutical anddiagnostic compositions for tumor immunotherapy and diagnosis,respectively. The use of the foregoing compositions in the preparationof a medicament is however preferred.

Hence, it is a particular object of the present invention to providemethods for treating or preventing a cancerous disease such as primarybreast carcinoma and metastases. The methods comprise administering aneffective concentration of an antibody or antibody derivative to thesubject where the antibody targets tumor tissue and cells.

Further embodiments of the present invention will be apparent from thedescription and Examples that follow. Furthermore, the description ofthe present invention, where necessary or appropriate, may besupplemented with the disclosure content of applicant's earlier Europeanpatent application EP 07 005 180.0 filed with the European Patent Officeon Mar. 13, 2007.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Memory B cell culture well 12 D7 contains antibodies specificfor NY-ESO-1. Medium conditioned by memory B cell cultures was assayedfor the presence of NY-ESO-1-specific human antibodies A) In ELISAdisplaying full length recombinant NY-ESO-1. B) In immunohistochemistryon NY-ESO-1-positive mamma carcinoma (mc) and on NY-ESO-1-negativecontrol tissue (ct). Shown is the staining obtained with conditionedmedium of two ELISA-positive memory B cell culture wells (9D1, 12D7). C)NY-ESO-1-specific antibody contained in well 12D7 is of the IgG1subclass as demonstrated by the staining of NY-ESO-1-positive tissuewith B cell conditioned medium from culture well12D7 followed bysecondary antibodies against IgG subclasses IgG1-4.

FIG. 2: Recombinant human antibody 12D7 clone number 4 obtained bysingle cell RT-PCR of cultured memory B cells specifically recognizesNY-ESO-1 in ELISA and on tissue sections. Supernatant fluid (SN)harvested from 293T HEK cells transfected with immunoglobulin heavy andlight chain expression vectors expressing clone 12D7 number four wastested for specificity to NY-ESO-1 in A) ELISA displaying full lengthNY-ESO-1. ELISA values are indicated for undiluted SN (1:12 D7.4 SN) a1/10 dilution (2:12 D7.4 SN) and a 1/100 dilution (3:12 D7.4 SN). Forcomparison, the ELISA signal obtained with plasma of the patient fromwhich the memory B cell cultures were derived used as a 1/100 dilutionis also shown (4). As controls, the absence of binding to NY-ESO-1coated ELISA plates of SN obtained upon transfection of an irrelevantrecombinant antibody produced in the same way as 12D7.4 is shown (5) aswell as the absence of binding of 12D7 clone No. 4 to ELISA platescoated with an irrelevant antigen. B) Immunohistochemistry onNY-ESO-1-positive mamma carcinoma (mc) and on NY-ESO-1-negative controltissue (ct) shows specific binding of recombinant 12D7 clone No. 4 tomamma carcinoma.

FIG. 3: Characteristics of human monoclonal antibody Manhattan. Epitopemapping was performed using overlapping peptides spanning the entireNY-ESO-1 protein coated onto ELISA plates. A) Manhattan specificallybinds to a peptide spanning amino acids 11 to 30 at the N-terminus ofthe NY-ESO-1 protein. B) serum of patient C1 recognizes various peptidefragments at the N-terminus and the mid-region of NY-ESO-1. C)Competition ELISA experiments with NY-ESO-1₁₁₋₃₀ peptide determine theavidity of Manhattan as KD=10⁻¹⁰. D) Immunofluorescence staining ofNY-ESO-1-positive cell line SK-MEL-37 with humAb Manhattan showsco-localization of NY-ESO-1 staining with nuclear marker Hoechst.Control antibody human recombinant 8-15c5 specific for MOG does notbind.

FIG. 4: Amino and nucleotide sequences of the variable region, i.e.heavy chain and kappa light chain of antibody 12D7. Complementaritydetermining regions (CDRs) are underlined.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to means and methods fordiscovering therapeutically efficient tumor (associated) antigen bindingmolecules, i.e. antibodies from clinically pre-selected cancer patients.In particular, antibodies and antigen-binding fragments thereof areprovided, which demonstrate the immunological binding characteristicsand/or biological properties as outlined for the antibody illustrated inthe Examples. Where present, the term “immunological bindingcharacteristics,” or other binding characteristics of an antibody withan antigen, in all of its grammatical forms, refers to the specificity,affinity, cross-reactivity, and other binding characteristics of anantibody.

Naturally, the present invention extends to the antibody producing celllines and recombinant cells as well. The present invention furtherrelates to diagnostic assays and kits that comprise the binding moleculeof the present invention and to therapeutic methods based thereon.

The present invention is based on a novel approach for the isolation andcloning of cancer patient-derived antibodies by using a method foridentifying, validating and producing tumor-specific diagnostically andtherapeutically useful binding molecules essentially as disclosed inapplicant's co-pending international application, serial numberPCT/EP2008/000053 “Method of providing disease-specific bindingmolecules and targets”, filed on Jan. 7, 2008, the disclosure content ofwhich is incorporated herein by reference. As demonstrated in theappended examples, a method for the isolation, molecular cloning andrecombinant production of patient-derived human antibodies totumor-associated antigens could be established. This is achieved by thescreening of oligoclonal memory B cell cultures established from patientperipheral blood lymphocytes (PBLs) combined with a molecular cloningstep using single cell RT-PCR and the re-screening of recombinantantibody clones with tissue micro-sections. In accordance with themethod of the present invention a human antibody specific for thetumor-associated antigen NY-ESO-1 was cloned from a melanoma patient whowas seropositive for NY-ESO-1 in ELISA and on autologous tumor sections.The screening was performed on ELISA and on tumor tissue using anadaptation of the tissue microarray technology. The obtainedtissue-reactive human monoclonal antibody was shown to bind to theN-terminus of NY-ESO-1 that is also shared by the tumor-associatedantigen LAGE-1; see Example 3.

This method greatly facilitates the cloning of patient-derivedantibodies; applied to selected clinical responders it is expected tolead to the identification and isolation of novel candidate antibodiesfor the immunotherapy of cancer as well as a means for the isolation ofnovel tumor (associated) antigens which because of the selectivity andspecificity of the method of the present invention may be more reliablefor use as tumor markers and targets for therapeutic intervention.

Accordingly, in a first aspect the present invention relates to a methodof isolating a human binding molecule specific for a tumor antigen ortumor-associated antigen comprising:

-   (a) subjecting a sample obtained from a patient bearing a tumor    antigen or tumor-associated antigen positive tumor wherein said    patient shows a at least partial clinical response or is    symptom-free to a specimen of tumor cells or tissue of predetermined    clinical characteristics; and-   (b) identifying and optionally isolating an antibody which binds to    said specimen but not to corresponding cells or tissues of a healthy    subject.

“Associated” is used herein because while it is clear that the relevantmolecule was specifically and aberrantly, respectively, expressed by atumor or cancer such as melanoma and breast, other cancers such asprostate and lung may also express the antigen and/or non-cancerouscells and tissue.

Unless stated otherwise, the terms “cancer” and “tumor” are usedinterchangeably herein.

For the sake of clarity only and without restricting the scope of thepresent invention most of the following embodiments are discussed withrespect to human antibodies and antibody-like molecules which representthe preferred binding molecules for the development of therapeutic anddiagnostic agents in accordance with the present invention. However, itis to be understood that as used in context of the present invention theterm “antibody”, and fragment thereof, may also refer to othernon-antibody binding molecules that bind to a human derived tumor(associated) antigen including but not limited to hormones, receptors,ligands, major histocompatibility complex (MHC) molecules, chaperonessuch as heat shock proteins (HSPs) as well as cell-cell adhesionmolecules such as members of the cadherin, integrin, C-type lectin andimmunoglobulin (Ig) superfamilies.

Whether a given structure, for example cell or tissue displays a tumor(associated) antigen can be verified by reversing the method describedbelow for isolating and characterizing a tumor (associated) antigenspecific binding molecule in that a binding molecule, for exampleantibody identified by said method is used to screen a sample forbinding to the antibody, thereby determining the presence of a tumor(associated) antigen.

The method of the present invention can be performed as outlined in theExamples section with means well known to a person skilled in the art.For example, a liquid sample obtained from the patient can be passedthrough a first aperture of a duct which is in contact with the specimentarget structure firmly held in an object holder, thereby allowingputative binding molecules present in the sample, either in a solubleform or expressed on the cell surface and membrane, respectively, tobind to said target structure. The liquid sample may contain for examplelymphocytes and/or antibodies while the specimen may be a tissue sectionor a membrane coated with molecules or molecular combinations which aredistinct for a pathological target structure.

Any non-binding matter can be removed via the second duct aperture. Atthe same time, the temperature of the object holder may be controlled byan object holder thermostat, for example at a temperature at whichnatural binding of the putative binding molecule to the tumor(associated) antigen specific for the specimen takes place in the humanbody. By way of the flowing motion, i.e. passing the liquid samplecontaining binding molecules, preferably at body temperature over thetarget structure natural systems of binding interactions can besimulated. However, other methods of incubating the sample with thespecimen such as by means of a shaker or rotating table may be used aswell. A particular advantage of the above-mentioned system is that itallows an interruption of metabolic processes at any time by decreasingthe temperature of the object holder by means of the object holderthermostat. In doing so, the temperature of the object holder can bedecreased to for example 2-10° C., in particular 4° C. A correspondingdevice that can be used in accordance with the method of the presentinvention is described in European patent application EP 1 069 431 A2.Hence, the method of the present invention will allow identification andcharacterization of the binding partners as well as at the same time toidentify and characterize the molecular classes, molecular groups and/ormolecular parts required for the binding process, i.e. the targetstructures of the specimen, which hitherto may be unknown. This will notonly open up new possible ways of diagnosis, but will also provide a newtest system for therapeutic approaches on a molecular level.

As a patient may qualify in accordance with the present invention a poolof healthy volunteers if specific tumor markers, genotype and/orcancerous phenotype predict a high probability of a status of a tumor,which has surprisingly—and possibly due to a specific endogenous immuneresponse—not become clinically manifest, however, by means of earlyintervention of the humoral immune system with or without involvement ofcellular components of the immune system.

In principle samples from patients may be used, who have undergone anactive immunization with a tumor (associated) antigen, wherein theantibody development has been boosted by the immunization. However,samples from volunteers which have not received such immunization orcorresponding tumor medication may be used as well.

According to the present invention, samples of a cancer patient, i.e. ofindividuals that have been clinically pre-selected are analyzed for thepresence of binding molecules specifically recognizing specimen ofpathologically conspicuous structures, for example in ex vivo tissuefrom clinico-pathologically characterized human patients or animalmodels like, for example, transgenic mice, or in vitro cell structures,or in pathological allogenic or xenogenic tissue. Preferably, saidpatient and/or as said subject providing the specimen are human, mostpreferably both. Preferably, said patient has been determined to beaffected with a not yet manifested tumor or at risk to develop a tumorby the presence or absence of a tumor marker, or by an unusually stableclinical course.

The characteristic cancerous altered sample, cell or tissue specimen ispreferably displayed by optical detection after reaction with a bindingmolecule, i.e. antibody of the present invention. The specimen may beobtained as/from a cell sample, tissue section, cellular smear test,cell or tissue sample of an animal model of a human tumor or in vitrocultured cell and tissue material. Histopathological grading can beperformed for example according the modified Bloom and Richardson system(Ellis et al., Tumors of the breast. In: Tavassoli F A, Devilee P.Genetics Tumors of the Breast and the Female Genital Organs. Lyon: IARCPress, (2003), 9-110). Preferably, the method of the present inventionemploys a multiple tissue microarray (TMA) as outlined in the Examples;for tissue microarray (TMA) technology comprising miniaturized pathologyarchives for high-throughput in situ studies see also, e.g., Bubendorfet al., J. Pathol., 195 (2001), 72-79, the disclosure content of whichis incorporated herein by reference. In particular, the method of thepresent invention preferably employs immunohistochemistry (IHC) usingtissue microarrays (TMA). For example, TMAs and large paraffin embeddedtissue sections can be analyzed with the Ventana Benchmark automatedstaining system (Ventana Medical Systems S.A., Illrich, CEDEX, France)using Ventana reagents for the entire procedure. Four-micron thickformalin-fixed, paraffin-embedded large tissue sections or TMA sectionscan be used. Paraffin sections are deparaffinized in xylene andrehydrated in decreasing concentrations of ethanol. Following a rinsewith distilled water endogenous peroxidase can be blocked using theVentana endogenous peroxidase blocking kit. For antigen retrieval,slides are heated with cell conditioning solution (CC1) in a standardprotocol. The human antibody candidate and sample containing the samecan be adjusted to the Ventana Benchmark system by performing titrationsand initial reactivity assessment, for example using spermatogonia ofthe testis as a positive control in case of supposed antiNY-ESO-1-antibodies. Antigen staining can usually be accepted aspositive when there is nuclear and/or cytoplasmic and/or cell surfacestaining. For statistical analysis, the immunohistochemical reactivityfor the tumor antigen and taa such as NY-ESO-1 can be measured aspercentage of positive cells per spot regardless of stainingintensities. For tables, staining can be categorized into positive(>/=5% positive cells) and negative (<5% positive cells). To determinethe impact of possible tumor antigen or taa expression heterogeneity,large paraffin sections from tumors with taa, e.g., NY-ESO-1 positivity(n=about 5 to 20) and negativity (n=about 5 to 20) on the TMA can beimmunohistochemically analyzed; see also the Examples. In addition, oralternatively, multiple tissue cores from a variety of patients andtumors can be used.

As mentioned, the sample to be analyzed may comprise a body fluid, acell sample or the supernatant of a cell sample or a derivative thereof.Body fluids such as plasma or peripheral blood can be collectedfollowing standard clinical procedures after informed consent of thepatients. Most preferably, the sample comprises or is derived fromB-cells or memory B-cells and/or comprises antibodies.

A particular approach in employing the method according to the presentinvention is testing samples of B cells and B memory cells fromclinically pre-selected volunteers against arrays of specimen of tumorconspicuous tissues expressing a differentiation and cancer testis (CT)antigen, respectively, selected from the group consisting of SCP-1,SSX-4, HOM-TES-85/CT-8, GAGE, SSX-1, SSX-2, NY-BR-1, LAGE-1 andNY-ESO-1. Differentiation and cancer testis (CT) antigen represent agroup of promising targets for immunotherapy, because of their exclusiveexpression in testicular germ cells and various malignancies (Scanlan etal., Immunol. Rev. 188 (2002), 22-32). An example for breast cancerspecific differentiation antigen is NY-BR-1, which is selectivelyexpressed in normal breast epithelium and about 60% of primary breastcancers on protein level making it a potential target for immunotherapy(Varga et al., Clin Cancer Res. 12 (2006), 2745-2751).

Over the past years, 44 different CT gene families have been identifiedwith 89 individual genes. In breast cancer, many CT antigens have beendescribed on mRNA level; SCP-1, SSX-4, HOM-TES-85/CT-8, GAGE, SSX-1 andNY-ESO-1 (Mischo et al., Int. J. Cancer 118 (2006), 696-703; Sugita etal., Cancer Res. 64 (2004), 2199-2204). NY-ESO-1 has been originallyidentified in an esophageal cancer patient using an antibody-basedcloning technique (SEREX, see supra). Recently it could be shown thatNY-ESO-1 may represent the most immunogenic CT antigen, becausespontaneous cellular and humoral immune responses can be observed in ahigh percentage of patients with NY-ESO-1 expressing tumors (Gnjatic etal., Proc. Natl. Acad. Sci. USA 100 (2003), 8862-8867; Jager and Knuth,Breast 14 (2005), 631-635).

Since CT antigens are selectively expressed in human tumor cells and inspermatogonias of the testis, they represent a promising group of targetantigens for an immunotherapeutic approach in cancer patients. Amongthem, NY-ESO-1 appears to be strongly immunogenic and is known to inducean efficient humoral and cellular immune response in patients withmelanoma and ovarian, breast, lung, as well as bladder cancer making itan ideal target for active cancer immunotherapy. Therefore, in aparticular preferred embodiment, the tumor conspicuous tissue employedin the method of the present invention expresses tumor-associatedantigen NY-ESO-1. For information on the nucleotide and amino acidsequences as well as origin, primary literature, etc. of tumor antigensand tumor associated antigens see appropriate databases such asUniProtKB/Swiss-Prot hosted by EMBL, in which an entry for, e.g.,NY-ESO-1 may be found under primary accession number P78358.

In a preferred embodiment of the method of the present invention saidspecimen comprises autologous tumor tissue or cells of said patient,most preferably said patient is a melanoma patient and said specimencomprises autologous sections derived from a lymph node metastasis; seealso the examples.

In a further embodiment, the method of the present invention furthercomprises the steps of:

-   (i) purifying B-cells or memory B-cells from a sample which has been    identified to contain binding molecules, i.e. antibodies which bind    to said specimen but not to corresponding cells or tissue of a    healthy subject;-   (ii) obtaining the immunoglobulin gene repertoire for said    antibodies from said B-cells or memory B-cells; and-   (iii) using said repertoire to express said antibodies, optionally    wherein step (ii) comprises the steps of:-   (iv) obtaining mRNA from said B-cells or memory B-cells;-   (v) obtaining cDNA from the mRNA of step (iv); and-   (vi) using a primer extension reaction to amplify from said cDNA the    fragments corresponding to the heavy chains (HC) and the kappa light    chains (LC) of said antibodies.

Methods of producing clones of an immortalized human B cell and B memorylymphocyte, comprising the step of transforming human B memorylymphocytes using Epstein Barr Virus (EBV) in the presence of apolyclonal B cell activator are summarized in international applicationWO2004/076677. This international application also describes methods forobtaining a nucleic acid sequence that encodes an antibody of interest,comprising the steps of preparing an immortalized B cell clone andobtaining/sequencing nucleic acid from the B cell clone that encodes theantibody of interest and further inserting the nucleic acid into orusing the nucleic acid to prepare an expression host that can expressthe antibody of interest, culturing or sub-culturing the expression hostunder conditions where the antibody of interest is expressed and,optionally, purifying the antibody of interest. It goes without sayingthat the nucleic acid may be manipulated in between to introducerestriction sites, to change codon usage, and/or to add or optimizetranscription and/or translation regulatory sequences. All thesetechniques are state of the art and can be performed by the personskilled in the art without undue burden.

However, since initial attempts at the cellular cloning of identifiedantigen-specific EBV-transformed human memory B cells had not beensuccessful, RT-PCR of single sorted cells is preferably employed forobtaining the immunoglobulin gene repertoire for said antibody; see alsothe Examples.

In a further aspect, the present invention relates to a binding moleculewhich is capable of selectively recognizing a tumor antigen ortumor-associated antigen, which preferably can be obtained or validatedby the method of the present invention described hereinbefore andillustrated in the Examples. In one preferred embodiment, the bindingmolecule of the present invention recognizes an antigen selected fromcancer testis (CT) antigens, such as selected from the group consistingof SCP-1, SSX-4, HOM-TES-85/CT-8, GAGE, SSX-1, SSX-2, NY-BR-1, LAGE-1and NY-ESO-1; see also supra. Most preferably, said antigen istumor-associated antigen NY-ESO-1.

NY-ESO-1 is a cancer testis antigen expressed in various malignanciesand testicular germ cells; see also supra. Because of its capacity toinduce specific humoral and cellular immunity in patients withNY-ESO-1-positive carcinomas, it represents a promising target forcancer immunotherapy. In a particularly preferred embodiment, saidantibody binds to an epitope defined by an amino acid sequence set forthin SEQ ID NO: 11 representing the amino acid residues 11 to 30 of theNY-ESO-1 protein. In this context it should however be understood thatwhile a tumor of the patient and the tumor tissue specimen,respectively, have been predetermined to express one or a set of tumor(associated) antigen(s), the antibody identified in accordance with themethod of the present invention to specifically bind to the tumor tissuespecimen but not to corresponding healthy tissue does not necessarilyrecognize the predetermined antigen but a different antigen, hithertounknown to be specifically or aberrantly expressed in tumor tissue.Therefore, the method of the present invention is also suitable toidentify and isolate novel tumor antigens and tumor associated antigens,respectively, and their cognate antibodies; see also infra.

Means and methods for the recombinant production of binding molecules,in particular antibodies and mimics thereof as well as methods ofscreening for competing binding molecules, which may or may not beantibodies, are known in the art; see also the Examples. However, asdescribed herein, in particular with respect to therapeutic applicationsin human the antibody of the present invention is a human antibody inthe sense that application of said antibody is substantially free of aHAMA response otherwise observed for chimeric and even humanizedantibodies.

Moreover, as demonstrated in appended Example 3, a binding molecule,i.e. antibody has been identified and cloned, which displaysparticularly high binding affinity with a equilibrium dissociationconstant (KD) of the interaction with its cognate antigen in the lowernanomolar range. Preferably, the binding affinity of the bindingmolecule of the present invention with its cognate antigen is about atleast 10⁻⁷M, more preferably at least 10⁻⁸M, particularly preferred10⁻⁹M and still more preferred at least 10⁻¹⁰M.

The present invention exemplifies such binding molecule, i.e. antibodyand binding fragments thereof, which may be characterized by comprisingin their variable region, i.e. binding domain at least onecomplementarity determining region (CDR) of the VH and/or VL of thevariable region comprising the amino acid sequence depicted in FIG. 4 of(V_(H)) (SEQ ID NO: 2) and (V_(L)) (SEQ ID NO: 4). An exemplary set ofCDRs of the above amino acid sequences of the V_(H) and/or V_(L) regionas depicted in FIG. 4 are given in SEQ ID NOs: 5 to 10. However, asdiscussed in the following the person skilled in the art is well awareof the fact that in addition or alternatively CDRs may be used, whichdiffer in their amino acid sequence from those set forth in SEQ ID NOs:5 to 10 by one, two, three or even more amino acids in case of CDR2 andCDR3.

Alternatively, the antibody of the present invention is an antibody orantigen-binding fragment thereof, which competes for binding to theNY-ESO-1 with at least one of the antibodies having the V_(H) and/orV_(L) region as depicted in FIG. 4. Those antibodies may be murine aswell, however, humanized, xenogeneic, or chimeric human-murineantibodies being preferred, in particular for therapeutic applications.An antigen-binding fragment of the antibody can be, for example, asingle chain Fv fragment (scFv), a F(ab′) fragment, a F(ab) fragment, oran F(ab′)₂ fragment.

For some applications only the variable regions of the antibodies arerequired, which can be obtained by treating the antibody with suitablereagents so as to generate Fab′, Fab, or F(ab″)₂ portions. Suchfragments are sufficient for use, for example, in immunodiagnosticprocedures involving coupling the immunospecific portions ofimmunoglobulins to detecting reagents such as radioisotopes.

As an alternative to obtaining immunoglobulins directly from the cultureof immortalized B cells or B memory cells, the immortalized cells can beused as a source of rearranged heavy chain and light chain loci forsubsequent expression and/or genetic manipulation. Rearranged antibodygenes can be reverse transcribed from appropriate mRNAs to produce cDNA.If desired, the heavy chain constant region can be exchanged for that ofa different isotype or eliminated altogether. The variable regions canbe linked to encode single chain Fv regions. Multiple Fv regions can belinked to confer binding ability to more than one target or chimericheavy and light chain combinations can be employed. Once the geneticmaterial is available, design of analogs as described above which retainboth their ability to bind the desired target is straightforward.Methods for the cloning of antibody variable regions and generation ofrecombinant antibodies are known to the person skilled in the art andare described, for example, Gilliland et al., Tissue Antigens 47 (1996),1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, however, mammaliancells are preferred. Typical mammalian cell lines useful for thispurpose include CHO cells, HEK 293 cells, or NSO cells. The productionof the antibody or analog is then undertaken by culturing the modifiedrecombinant host under culture conditions appropriate for the growth ofthe host cells and the expression of the coding sequences. Theantibodies are then recovered by isolating them from the culture. Theexpression systems are preferably designed to include signal peptides sothat the resulting antibodies are secreted into the medium; however,intracellular production is also possible.

As already mentioned, the method of the present invention can beextended to also identify and isolate the cognate antigen bound by theantibody of the present invention identified and cloned beforehand.Thus, in one embodiment the method of the present invention serves andis used for the identification and isolation of novel tumor (associated)antigens.

One key methodology aiming at the achieving the substantially the samegoal is described by Sahin, et al., Proc. Natl. Acad. Sci. USA 92:11810-11913 (1995), incorporated by reference herein; see also, see U.S.Pat. Nos. 5,698,396, and 6,252,052 both of these references areincorporated by reference herein. To summarize, the method involves theexpression of cDNA libraries in a prokaryotic host, said libraries beingsecured from a tumor sample. The expressed libraries are thenimmuno-screened with absorbed and diluted sera, in order to detect thoseantigens which elicit high titer humoral responses. This methodology isknown as the SEREX method (“Serological identification of antigens byRecombinant Expression Cloning”). The methodology has been employed toconfirm expression of previously identified tumor-associated antigens,as well as to detect new ones; see the above referenced patentapplications and Sahin et al., supra, as well as Crew et al., EMBO J.144 (1995), 2333-2340.

In contrast, in accordance with the method of the present inventionserological identification of tumor-specific antigens is accomplished byuse of a cognate antibody that has been screened against tumor tissuederived from cancer patients who show a clinical response and havedeveloped antibodies against their autologous tumor tissue. The antigenand truncated forms thereof can than be validated to be reactive withantibodies in the serum of said cancer patient and other cancerpatients.

Though at first glance the conventional SEREX method seems to be easierto perform as no cloning of B cells and antibody is needed, it isprudent to expect that the method of the present invention is moresensitive and reliable than the SEREX methodology. Besides, it is ofcourse advantageous to be provided with both a specific tumor markerprotein as well as a corresponding antibody which have been clinicallyproved to be of diagnostic relevance at least.

Thus, once the target structure, i.e. the tumor-associated antigen hasbeen tagged by the sample and respective binding molecule therein, itmay be identified and its encoding nucleic acid molecule isolated andcloned. Identification of the amino acid sequence of the antigen can beachieved by means and methods well known in the art, for example usingmass spectrometric (MS) techniques such as those described ininternational application WO00/11208 and specifically those described inHock et al., Nat Med 8 (2002), 1270-1275; Hock et al., Neuron 38 (2003),547-554. Thus, in case the antibody identified in accordance withpresent invention produced in vitro binds to pathological structures,for example to tumor tissue sections, but not significantly to healthytissues, a promising antibody candidate has been identified whosemolecular target structure can subsequently be enriched and purified viaits binding properties to the antibody from pathological tissues and, asa result, can be identified and characterized by means of proteinanalytical and mass spectrometric methods like, for example, MALDI/TOF(Williams, Methods Cell. Biol. 62 (2000), 449-453; Yates, J. Mass.Spectrom. 33 (1998), 1-19).

Accordingly, in another embodiment the present invention relates to atumor antigen and tumor-associated antigen, respectively, which isrecognized by the binding molecule, especially antibody of the presentinvention described hereinbefore, both in peptide form and in posttranslational modified form, wherein the antigen is preferably a peptideconsisting of least 6-50, and preferably no more than 10-100 amino acidsin length, which contain the cognate epitope. Most preferably, theantigen of the present invention comprises the amino acid sequence ofSEQ ID NO: 11 and consists of about 10 to 30 amino acids, and preferablyis no more than about 20 amino acids in length. The molecule is largeenough to be antigenic without any posttranslational modification, andhence it is useful as an immunogen, when combined with an adjuvant (orwithout it), in both precursor and post-translationally modified forms.These antigens and peptides can be used to determine whether or notantibodies are present in a sample, such as serum or blood. Preferably,the antigen of the present invention is capable of eliciting a humoralresponse in human. In particular, the present invention relates to noveltumor antigens and tumor-associated antigens detectable with theantibody of the present invention, which hitherto have not beendisclosed in the prior art and/or subject of a patent or patentapplication.

In accordance with the above, the present invention also relates to apolynucleotide encoding the antigen or binding molecule of the presentinvention, in case of the antibody preferably at least a variable regionof an immunoglobulin chain of the antibody described above. Typically,said variable region encoded by the polynucleotide comprises at leastone complementarity determining region (CDR) of the V_(H) and/or V_(L)of the variable region of the said antibody. The person skilled in theart knows that each variable domain (the heavy chain V_(H) and lightchain V_(L)) of an antibody comprises three hypervariable regions,sometimes called complementarity determining regions or “CDRs” flankedby four relatively conserved framework regions or “FRs” and refer to theamino acid residues of an antibody which are responsible forantigen-binding. The hypervariable regions or CDRs of the human IgGsubtype of antibody comprise amino acid residues from residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain as described by Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and/or those residues from ahypervariable loop, i.e. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3)in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101(H3) in the heavy chain variable domain as described by Chothia et al.,J. Mol. Biol. 196 (1987), 901-917. Framework or FR residues are thosevariable domain residues other than and bracketing the hypervariableregions. The term “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with a dissociationconstant (K_(D)) of 10⁻⁷ M or less, and binds to the predeterminedantigen with a K_(D) that is at least twofold less than its K_(D) forbinding to a nonspecific antigen (e.g., BSA, casein, or any otherspecified polypeptide) other than the predetermined antigen. The phrases“an antibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”. As used herein “highlyspecific” binding means that the relative K_(D) of the antibody for thespecific target epitope, i.e. tumor antigen or taa is at least 10-foldless than the K_(D) for binding that antibody to other ligands.Preferably, the antibody binds its cognate tumor antigen and taa,respectively, with a dissociation constant (K_(D)) of 10⁻⁹ M or less.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method; see, for example, Berzofsky etal., “Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W.E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H.Freeman and Company New York, N Y (1992), and methods described herein.The measured affinity of a particular antibody-antigen interaction canvary if measured under different conditions, e.g., salt concentration,pH. Thus, measurements of affinity and other antigen-binding parameters,e.g., K sub D, IC50, are preferably made with standardized solutions ofantibody and antigen, and a standardized buffer.

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art willreadily appreciate that using the variable domains or CDRs describedherein antibodies can be constructed according to methods known in theart, e.g., as described in European patent applications EP 0 451 216 A1and EP 0 549 581 A1. Furthermore, the person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat. Thus, the present invention also relates to antibodieswherein one or more of the mentioned CDRs comprise one or more,preferably not more than two amino acid substitutions. Preferably, theantibody of the invention comprises in one or both of its immunoglobulinchains two or all three CDRs of the variable regions as set forth in SEQID NOs: 5 to 10.

The polynucleotide of the invention encoding the above describedantibody may be, e.g., DNA, cDNA, RNA or synthetically produced DNA orRNA or a recombinantly produced chimeric nucleic acid moleculecomprising any of those polynucleotides either alone or in combination.Preferably said polynucleotide is part of a vector. Such vectors maycomprise further genes such as marker genes which allow for theselection of said vector in a suitable host cell and under suitableconditions.

Preferably, the polynucleotide of the invention is operatively linked toexpression control sequences allowing expression in prokaryotic oreukaryotic cells. Expression of said polynucleotide comprisestranscription of the polynucleotide into a translatable mRNA. Regulatoryelements ensuring expression in eukaryotic cells, preferably mammaliancells, are well known to those skilled in the art. They usually compriseregulatory sequences ensuring initiation of transcription and optionallypoly-A signals ensuring termination of transcription and stabilizationof the transcript. Additional regulatory elements may includetranscriptional as well as translational enhancers, and/or naturallyassociated or heterologous promoter regions.

In this respect, the person skilled in the art will readily appreciatethat the polynucleotides encoding at least the variable domain of thelight and/or heavy chain may encode the variable domains of bothimmunoglobulin chains or only one. Likewise, said polynucleotides may beunder the control of the same promoter or may be separately controlledfor expression. Possible regulatory elements permitting expression inprokaryotic host cells comprise, e.g., the P_(L), lac, trp or tacpromoter in E. coli, and examples for regulatory elements permittingexpression in eukaryotic host cells are the AOX1 or GAL1 promoter inyeast or the CMV-, SV40-, RSV-promoter, CMV-enhancer, SV40-enhancer or aglobin intron in mammalian and other animal cells.

Beside elements which are responsible for the initiation oftranscription such regulatory elements may also comprise transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Furthermore, depending on theexpression system used leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediummay be added to the coding sequence of the polynucleotide of theinvention and are well known in the art. The leader sequence(s) is (are)assembled in appropriate phase with translation, initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein, or a portion thereof, intothe periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including a C- orN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct. In this context, suitable expression vectors are known in theart such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia),pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (GIBCO BRL).

Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells, but control sequences for prokaryotic hosts may also beused. Once the vector has been incorporated into the appropriate host,the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the immunoglobulin light chains, heavy chains,light/heavy chain dimers or intact antibodies, binding fragments orother immunoglobulin forms may follow; see, Beychok, Cells ofImmunoglobulin Synthesis, Academic Press, N.Y., (1979).

Furthermore, the present invention relates to vectors, particularlyplasmids, cosmids, viruses and bacteriophages used conventionally ingenetic engineering that comprise a polynucleotide encoding the antigenor preferably a variable domain of an immunoglobulin chain of anantibody of the invention; optionally in combination with apolynucleotide of the invention that encodes the variable domain of theother immunoglobulin chain of the antibody of the invention. Preferably,said vector is an expression vector and/or a gene transfer or targetingvector. Expression vectors derived from viruses such as retroviruses,vaccinia virus, adeno-associated virus, herpes viruses, or bovinepapilloma virus, may be used for delivery of the polynucleotides orvector of the invention into targeted cell population. Methods which arewell known to those skilled in the art can be used to constructrecombinant viral vectors; see, for example, the techniques described inSambrook, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory (1989) N.Y. and Ausubel, Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y.(1994). Alternatively, the polynucleotides and vectors of the inventioncan be reconstituted into liposomes for delivery to target cells. Thevectors containing the polynucleotides of the invention (e.g., the heavyand/or light variable domain(s) of the immunoglobulin chains encodingsequences and expression control, sequences) can be transferred into thehost cell by well known methods, which vary depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts; see Sambrook,supra.

The present invention furthermore relates to host cells transformed witha polynucleotide or vector of the invention. Said host cell may be aprokaryotic or eukaryotic cell. The polynucleotide or vector of theinvention which is present in the host cell may either be integratedinto the genome of the host cell or it may be maintainedextrachromosomally. The host cell can be any prokaryotic or eukaryoticcell, such as a bacterial, insect, fungal, plant, animal or human cell.Preferred fungal cells are, for example, those of the genusSaccharomyces, in particular those of the species S. cerevisiae. Theterm “prokaryotic” is meant to include all bacteria which can betransformed or transfected with a DNA or RNA molecules for theexpression of an antibody of the invention or the correspondingimmunoglobulin chains. Prokaryotic hosts may include gram negative aswell as gram positive bacteria such as, for example, E. coli, S.typhimurium, Serratia marcescens and Bacillus subtilis. The term“eukaryotic” is meant to include yeast, higher plant, insect andpreferably mammalian cells, most preferably HEK 293, NSO and CHO cells.Depending upon the host employed in a recombinant production procedure,the antibodies or immunoglobulin chains encoded by the polynucleotide ofthe present invention may be glycosylated or may be non-glycosylated.Antibodies of the invention or the corresponding immunoglobulin chainsmay also include an initial methionine amino acid residue. Apolynucleotide of the invention can be used to transform or transfectthe host using any of the techniques commonly known to those of ordinaryskill in the art. Furthermore, methods for preparing fused, operablylinked genes and expressing them in, e.g., mammalian cells and bacteriaare well-known in the art (Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).The genetic constructs and methods described therein can be utilized forexpression of the antibody of the invention or the correspondingimmunoglobulin chains in eukaryotic or prokaryotic hosts. In general,expression vectors containing promoter sequences which facilitate theefficient transcription of the inserted polynucleotide are used inconnection with the host. The expression vector typically contains anorigin of replication, a promoter, and a terminator, as well as specificgenes which are capable of providing phenotypic selection of thetransformed cells. Suitable source cells for the DNA sequences and hostcells for immunoglobulin expression and secretion can be obtained from anumber of sources, such as the American Type Culture Collection(“Catalogue of Cell Lines and Hybridomas,” Fifth edition (1985)Rockville, Md., U.S.A., which is incorporated herein by reference).Furthermore, transgenic animals, preferably mammals, comprising cells ofthe invention may be used for the large scale production of the antibodyof the invention.

Thus, in a further embodiment, the present invention relates to a methodfor the production of an antigen of the present invention or of a tumorantigen and taa-specific binding molecule, respectively, an antibody ora binding fragment or immunoglobulin chain(s) thereof, said methodcomprising

-   (a) culturing a cell as described above; and-   (b) isolating said antigen, binding molecule, antibody or binding    fragment or immunoglobulin chain(s) thereof from the culture.

The transformed hosts can be grown in fermentors and cultured accordingto techniques known in the art to achieve optimal cell growth. Onceexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like; see, Scopes, “Protein Purification”,Springer Verlag, N.Y. (1982). The antibody or its correspondingimmunoglobulin chain(s) of the invention can then be isolated from thegrowth medium, cellular lysates, or cellular membrane fractions. Theisolation and purification of the, e.g., recombinantly expressedantibodies or immunoglobulin chains of the invention may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies directed, e.g., against theconstant region of the antibody of the invention. It will be apparent tothose skilled in the art that the antibodies of the invention can befurther coupled to other moieties for, e.g., drug targeting and imagingapplications. Such coupling may be conducted chemically after expressionof the antibody or antigen to site of attachment or the coupling productmay be engineered into the antibody or antigen of the invention at theDNA level. The DNAs are then expressed in a suitable host system, andthe expressed proteins are collected and renatured, if necessary.

Substantially pure immunoglobulins of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity mostpreferred, for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the antibodies may then be used therapeutically(including extracorporally) or in developing and performing assayprocedures.

The present invention also involves a method for producing cells capableof expressing an antibody of the invention or its correspondingimmunoglobulin chain(s) comprising genetically engineering cells withthe polynucleotide or with the vector of the invention. The cellsobtainable by the method of the invention can be used, for example, totest the interaction of the antibody of the invention with its antigen.

As mentioned before, the immunoglobulin or its encoding cDNAs may befurther modified. Thus, in a further embodiment the method of thepresent invention comprises any one of the step(s) of producing achimeric antibody, humanized antibody, single-chain antibody,Fab-fragment, bi-specific antibody, fusion antibody, labeled antibody oran analog of any one of those. Corresponding methods are known to theperson skilled in the art and are described, e.g., in Harlow and Lane“Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988.When derivatives of said antibodies are obtained by the phage displaytechnique, surface plasmon resonance as employed in the BIAcore systemcan be used to increase the efficiency of phage antibodies which bind tothe same epitope as that of any one of the antibodies described herein(Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.Immunol. Methods 183 (1995), 7-13). The production of chimericantibodies is described, for example, in international applicationWO89/09622. Methods for the production of humanized antibodies aredescribed in, e.g., European application EP-A1 0 239 400 andinternational application WO90/07861. Further sources of antibodies tobe utilized in accordance with the present invention are so-calledxenogeneic antibodies. The general principle for the production ofxenogeneic antibodies such as human antibodies in mice is described in,e.g., international applications WO91/10741, WO94/02602, WO96/34096 andWO 96/33735. As discussed above, the antibody of the invention may existin a variety of forms besides complete antibodies; including, forexample, Fv, Fab and F(ab)₂, as well as in single chains; see e.g.international application WO88/09344.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus; see, e.g., international applicationWO00/30680 for corresponding technical details.

Additionally, the present invention encompasses small peptides includingthose containing a binding molecule as described above, for examplecontaining the CDR3 region of the variable region of any one of thementioned antibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that heavy chain CDR3 (HCDR3) is the regionhaving a greater degree of variability and a predominant participationin antigen-antibody interaction. Such peptides may easily be synthesizedor produced by recombinant means to produce a binding agent usefulaccording to the invention. Such methods are well known to those ofordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can be produced by recombinant techniques by incorporatingthe DNA expressing the peptide into an expression vector andtransforming cells with the expression vector to produce the peptide.

Hence, the present invention relates to any binding molecule, antibodyor binding fragment which are obtainable in accordance with abovedescribed means and display the mentioned properties, i.e. whichspecifically recognize a tumor antigen or taa, and which for therapeuticuse preferably maintain a substantially human framework so as to bedevoid of immunogenicity in a patient. Such antibodies and bindingmolecules can be tested for their binding specificity and affinity byfor example by using the method of isolating human tumor (associated)antigen specific binding molecules described hereinbefore.

In a further embodiment of the present invention, the binding molecule,antibody, immunoglobulin chain or a binding fragment thereof or theantigen is detectably labeled. Labeling agents can be coupled eitherdirectly or indirectly to the antibodies or antigens of the invention.One example of indirect coupling is by use of a spacer moiety.Furthermore, the antibodies of the present invention can comprise afurther domain, said domain being linked by covalent or non-covalentbonds. The linkage can be based on genetic fusion according to themethods known in the art and described above or can be performed by,e.g., chemical cross-linking as described in, e.g., internationalapplication WO94/04686. The additional domain present in the fusionprotein comprising the antibody of the invention may preferably belinked by a flexible linker, advantageously a polypeptide linker,wherein said polypeptide linker comprises plural, hydrophilic,peptide-bonded amino acids of a length sufficient to span the distancebetween the C-terminal end of said further domain and the N-terminal endof the antibody of the invention or vice versa. The therapeutically ordiagnostically active agent can be coupled to the antibody of theinvention or an antigen-binding fragment thereof by various means. Thisincludes, for example, single-chain fusion proteins comprising thevariable regions of the antibody of the invention coupled by covalentmethods, such as peptide linkages, to the therapeutically ordiagnostically active agent. Further examples include molecules whichcomprise at least an antigen-binding fragment coupled to additionalmolecules covalently or non-covalently include those in the followingnon-limiting illustrative list. Traunecker, Int. J. Cancer Surp. SuDP 7(1992), 51-52, describe the bispecific reagent janusin in which the Fvregion directed to CD3 is coupled to soluble CD4 or to other ligandssuch as OVCA and IL-7. Similarly, the variable regions of the antibodyof the invention can be constructed into Fv molecules and coupled toalternative ligands such as those illustrated in the cited article.Higgins, J. Infect Disease 166 (1992), 198-202, described ahetero-conjugate antibody composed of OKT3 cross-linked to an antibodydirected to a specific sequence in the V3 region of GP120. Suchhetero-conjugate antibodies can also be constructed using at least thevariable regions contained in the antibody of the invention methods:Additional examples of specific antibodies include those described byFanger, Cancer Treat. Res. 68 (1993), 181-194 and by Fanger, Crit. Rev.Immunol. 12 (1992), 101-124. Conjugates that are immunotoxins includingconventional antibodies have been widely described in the art. Thetoxins may be coupled to the antibodies by conventional couplingtechniques or immunotoxins containing protein toxin portions can beproduced as fusion proteins. The antibodies of the present invention canbe used in a corresponding way to obtain such immunotoxins. Illustrativeof such immunotoxins are those described by Byers, Seminars Cell. Biol.2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.

The above described fusion protein may further comprise a cleavablelinker or cleavage site for proteinases. These spacer moieties, in turn,can be either insoluble or soluble (Diener et al., Science 231 (1986),148) and can be selected to enable drug release from the antigen at thetarget site. Examples of therapeutic agents which can be coupled to theantibodies and antigens of the present invention for immunotherapy aredrugs, radioisotopes, lectins, and toxins. The drugs with which can beconjugated to the antibodies and antigens of the present inventioninclude compounds which are classically referred to as drugs such asmitomycin C, daunorubicin, and vinblastine. In using radioisotopicallyconjugated antibodies or antigens of the invention for, e.g., tumorimmunotherapy, certain isotopes may be more preferable than othersdepending on such factors as leukocyte distribution as well as stabilityand emission. Depending on the autoimmune response, some emitters may bepreferable to others. In general, α and β particle emittingradioisotopes are preferred in immunotherapy. Preferred are short range,high energy a emitters such as ²¹²Bi. Examples of radioisotopes whichcan be bound to the antibodies or antigens of the invention fortherapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹²At, ²¹¹Pb,⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re. Other therapeutic agents which can be coupled tothe antibody or antigen of the invention, as well as ex vivo and in vivotherapeutic protocols, are known, or can be easily ascertained, by thoseof ordinary skill in the art. Wherever appropriate the person skilled inthe art may use a polynucleotide of the invention encoding any one ofthe above described antibodies, antigens or the corresponding vectorsinstead of the proteinaeous material itself.

Hence, the biological activity of the binding molecules, e.g. antibodiesidentified here suggests that they have sufficient affinity to make thempotential candidates for drug localization to cells expressing theappropriate surface structures of the diseased cell and tissue,respectively. This targeting and binding to cells could be useful forthe delivery of therapeutically or diagnostically active agents and genetherapy/gene delivery. Molecules/particles with an antibody of theinvention would bind specifically to cells/tissues expressing the tumorantigen or taa, and therefore could have diagnostic and therapeutic use.Thus, the antibody or the antigen of the present invention can belabeled (e.g., fluorescent, radioactive, enzyme, nuclear magnetic, heavymetal) and used to detect specific targets in vivo or in vitro including“immunochemistry” like assays in vitro. In vivo they could be used in amanner similar to nuclear medicine imaging techniques to detect tissues,cells, or other material expressing the tumor (associated) antigen.Thus, in a further embodiment the present invention relates to the useof a binding molecule or an antibody of the present invention or bindingfragment thereof for the preparation of a composition for in vivodetection of or targeting a therapeutic and/or diagnostic agent to atumor.

Moreover, the present invention relates to compositions comprising theaforementioned binding molecule, antibody or binding fragment or antigenof the present invention or chemical derivatives thereof, or thepolynucleotide, vector or cell of the invention. The composition of thepresent invention may further comprise a pharmaceutically acceptablecarrier. The term “chemical derivative” describes a molecule thatcontains additional chemical moieties that are not normally a part ofthe base molecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Furthermore, the pharmaceuticalcomposition of the present invention may comprise further anti-tumoragents such as interleukins or interferons depending on the intended useof the pharmaceutical composition. Hence, in a particular preferredembodiment the present invention relates to the use of the bindingmolecule, antibody or binding fragment of the present invention or of abinding molecule having substantially the same binding specificities ofany one thereof, the antigen, the polynucleotide, the vector or the cellof the present invention for the preparation of a pharmaceutical ordiagnostic composition for the preparation of a pharmaceutical ordiagnostic composition for treating or preventing the progression of atumor; for the amelioration of symptoms associated with a tumor; fordiagnosing or screening a subject for the presence of a tumor or fordetermining a subject's risk for developing a tumor. Said pharmaceuticalcomposition can be designed to be administered intravenously,intramuscularly, subcutaneously, intraperitoneally, intranasally,parenterally or as an aerosol; see also infra.

Hence, in one embodiment the present invention relates to a method oftreating or preventing the progression of a tumor in a subject; forameliorating the symptoms associated with a tumor; for diagnosing orscreening a subject for the presence of a tumor or for determining asubject's risk for developing a tumor, which method comprisesadministering to said subject an effective amount of any one of theafore-described binding molecules, antibodies, antigens,polynucleotides, vectors or cells of the instant invention. Inparticular, the therapeutic and diagnostic applications in accordancewith the present invention include melanoma and breast cancer, and aremost suitable for use in targeting a tumor comprising primary breastcarcinoma and/or metastases. Unless stated otherwise, the terms “tumor”,“cancer”, “carcinoma” and the like are used interchangeably herein.

Hence, the present invention encompasses any use of a tumor antigenbinding molecule comprising at least one CDR of the above describedhuman antibody, in particular for diagnosing and/or treating a disorderrelated to a tumor. Preferably, said binding molecule is an antibody ofthe present invention or an immunoglobulin chain thereof. In addition,the present invention relates to anti-idiotypic antibodies of any one ofthe mentioned antibodies described hereinbefore. These are antibodies orother binding molecules which bind to the unique antigenic peptidesequence located on an antibody's variable region near the antigenbinding site.

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described binding molecules,antibodies, antigen-binding fragments, polynucleotides, vectors or cellsof the invention and optionally suitable means for detection such asreagents conventionally used in immuno or nucleic acid based diagnosticmethods.

The antibodies of the invention are, for example, suited for use inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. Examples of immunoassays which can utilize theantibody of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA), the sandwich (immunometricassay), flow cytometry and the Western blot assay. The antigens andantibodies of the invention can be bound to many different carriers andused to isolate cells specifically bound thereto. Examples of well knowncarriers include glass, polystyrene, polyvinyl chloride, polypropylene,polyethylene, polycarbonate, dextran, nylon, amyloses, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble or insoluble for thepurposes of the invention. There are many different labels and methodsof labeling known to those of ordinary skill in the art. Examples of thetypes of labels which can be used in the present invention includeenzymes, radioisotopes, colloidal metals, fluorescent compounds,chemiluminescent compounds, and bioluminescent compounds; see also theembodiments discussed hereinabove.

By a further embodiment, the binding molecules, in particular antibodiesof the present invention may also be used in a method for the diagnosisof a tumor in an individual by obtaining a body fluid sample from thetested individual which may be a blood sample, a lymph sample or anyother body fluid sample and contacting the body fluid sample with anantibody of the instant invention under conditions enabling theformation of antibody-antigen complexes. The level of such complexes isthen determined by methods known in the art, a level significantlyhigher than that formed in a control sample indicating the tumor in thetested individual. In the same manner, the specific antigen bound by theantibodies of the invention may also be used. Thus, the presentinvention relates to an in vitro immunoassay comprising the antibody orthe antigen of the invention. A preferred embodiment of the presentinvention relates to the determination of cancer, melanoma and breastcancer in particular. The methods involve assaying for members of theso-called “cancer-testis” or “CT” antigen family, most preferablyNY-ESO-1.

In one embodiment, the present invention relates to a method fordetermining status of a cancerous condition, e.g., regression,progression of onset of a cancerous condition in a patient with a tumorthat expresses a tumor (associated) antigen such as NY-ESO-1, comprisingassaying a sample taken from said patient for antibodies whichspecifically bind to said antigen, and comparing a value obtained to aprior value obtained following assay of a prior sample taken from saidpatient, any difference there between being indicative of a change instatus of said cancerous condition. A corresponding method that can beemployed in accordance with the present invention is disclosed ininternational application WO01/07917. Alternatively, such method may beperformed with an antibody of the present invention.

In another embodiment, the present invention relates to a method fordetermining cancer cells, e.g., breast cancer cells in a samplecomprising assaying said sample for expression of at least one tumorassociated antigen like NY-ESO-1 by assaying for presence of the antigenprotein, with an antibody of the present invention which specificallybinds to said antigen, wherein expression of at least one of saidantigens is indicative of the presence of cancer cells in said sample. Asimilar method which may be adapted in accordance with the presentinvention is described in U.S. Pat. No. 6,338,947 for SCP-1, NY-ESO-1and SSX-2. Thus, the method of the present invention may furthercomprise assaying said sample for at least one of SCP-1, NY-ESO-1,SSX-1, SSX-2, SSX-4, MAGE-1, GAGE, MAGE-3, and LAGE-1.

In this context, the present invention also relates to meansspecifically designed for this purpose. For example, a protein- orantibody-based array may be used, which is for example loaded witheither antigens derived from the mentioned disorder-associated proteinand containing the tumor-associated antigen in order to detectautoantibodies which may be present in patients suffering from a tumor,in particular metastases, or with antibodies or equivalentantigen-binding molecules of the present invention which specificallyrecognize any one of those tumor-associated antigens. Design ofmicroarray immunoassays is summarized in Kusnezow et al., Mol. CellProteomics 5 (2006), 1681-1696. Accordingly, the present invention alsorelates to microarrays loaded with binding molecules or antigensidentified in accordance with the present invention.

The present invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, i.e. binding molecule,antibody or binding fragment thereof, antigen, polynucleotide, vector orcell of the present invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition or alternatively the kit comprisesreagents and/or instructions for use in appropriate diagnostic assays.The composition, i.e. kit of the present invention is of courseparticularly suitable for the diagnosis, prevention and treatment of adisorder which is accompanied with the presence of a tumor-associatedantigen defined above, in particular applicable for the treatment oftumors as mentioned above.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of partially or completely curing a disease and/oradverse effect attributed to the disease. The term “treatment” as usedherein covers any treatment of a disease in a mammal, particularly ahuman, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e. arresting itsdevelopment; or (c) relieving the disease, i.e. causing regression ofthe disease. Furthermore, the term “subject” or “patient” refers to amammal, preferably a human, in need of treatment for a condition,disorder or disease.

The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intra-muscular, topical orintradermal administration. Aerosol formulations such as nasal sprayformulations include purified aqueous or other solutions of the activeagent with preservative agents and isotonic agents. Such formulationsare preferably adjusted to a pH and isotonic state compatible with thenasal mucous membranes. Formulations for rectal or vaginaladministration may be presented as a suppository with a suitablecarrier.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the regimen as a regularadministration of the pharmaceutical composition should be in the rangeof 1 μg to 10 mg units per day. If the regimen is a continuous infusion,it should also be in the range of 1 μg to 10 mg units per kilogram ofbody weight per minute, respectively. Progress can be monitored byperiodic assessment. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as anti-tumor agents andcytotoxic drugs, depending on the intended use of the pharmaceuticalcomposition. Furthermore, the pharmaceutical composition may also beformulated as a vaccine, for example, if the pharmaceutical compositionof the invention comprises an antibody of the present invention forpassive immunization or tumor (associated) antigen for activeimmunization. Vaccine formulations for the treatment of cancer antigensemploying tumor associated antigens such as NY-ESO-1 are described forexample in international application WO2005/105139.

In addition, co-administration or sequential administration of otheragents may be desirable. A therapeutically effective dose or amountrefers to that amount of the active ingredient sufficient to amelioratethe symptoms or condition. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED50 (the dosetherapeutically effective in 50% of the population) and LD50 (the doselethal to 50% of the population). The dose ratio between therapeutic andtoxic effects is the therapeutic index, and it can be expressed as theratio, LD50/ED50. Preferably, the therapeutic agent in the compositionis present in an amount sufficient to prevent metastasis and neoplasticgrowth of cells.

The pharmaceutical compositions in accordance with the present inventioncan be used for the treatment of tumors and cancer including but notlimited to melanoma, primary breast cancer, hepatocellular carcinoma andmetastases as well as other human sarcomas and carcinomas, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, carcinoma of the head/neck, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, glioblastoma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocyticleukemia and acute myelocytic leukemia (myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia); chronic leukemia(chronic myelocytic (granulocytic) leukemia and chronic lymphocyticleukemia); and polycythemia vera, lymphoma (Hodgkin's disease andnon-Hodgkin's disease), multiple myeloma, and heavy chain disease.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Unlessotherwise stated, a term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations may be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this application andmanufacturer's specifications, instructions, etc) are hereby expresslyincorporated by reference; however, there is no admission that anydocument cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES

The examples which follow further illustrate the invention, but shouldnot be construed to limit the scope of the invention in any way.Detailed descriptions of conventional methods, such as those employedherein can be found in the cited literature; see also “The Merck Manualof Diagnosis and Therapy” Seventeenth Ed. ed by Beers and Berkow (Merck& Co., Inc. 2003).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. For furtherelaboration of general techniques useful in the practice of thisinvention, the practitioner can refer to standard textbooks and reviewsin cell biology and tissue culture; see also the references cited in theexamples. General methods in molecular and cellular biochemistry can befound in such standard textbooks as Molecular Cloning: A LaboratoryManual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); ShortProtocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley& Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985);Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization(Hames and Higgins eds. 1984); Transcription And Translation (Hames andHiggins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss,Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller andCalos, eds.); Current Protocols in Molecular Biology and Short Protocolsin Molecular Biology, 3rd Edition (Ausubel et al., eds.); andRecombinant DNA Methodology (Wu, ed., Academic Press). Gene TransferVectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold SpringHarbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.,eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In CellAnd Molecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir andBlackwell, eds., 1986). Protein Methods (Bollag et al., John Wiley &Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. eds.,Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., AcademicPress 1995); Immunology Methods Manual (Lefkovits ed., Academic Press1997); and Cell and Tissue Culture: Laboratory Procedures inBiotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents,cloning vectors and kits for genetic manipulation referred to in thisdisclosure are available from commercial vendors such as BioRad,Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.General techniquesin cell culture and media collection are outlined in Large ScaleMammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997),148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); LargeScale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375);and Suspension Culture of Mammalian Cells (Birch et al., BioprocessTechnol. 19 (1990), 251); Extracting information from cDNA arrays,Herzel et al., CHAOS 11 (2001), 98-107.

Supplementary Methods Patient Material

Tumor material as well as normal tissue not necessary for the routinehistopathological analysis was frozen in liquid nitrogen. Serum andblood for the isolation of memory B cells was collected from patient C1in accordance with the informed consent that was approved by the localEthical committee and signed by the patient.

Memory B Cell Culture

Memory B cells were isolated from human peripheral blood lymphocytes bya two step selection procedure. The pan B cell marker CD22 was used forthe positive selection of B cells using the MACS technology (Miltenyi,Bergisch Gladbach, Germany). PBL were labeled using MACS-conjugated antihuman CD22 mAbs, phycoerythrin-conjugated mAbs anti human IgD andAPC-conjugated antibodies anti human IgM, IgA, CD3, CD8, CD56 (BectonDickinson, Basel, Switzerland). Pan B cells were isolated by positiveselecting CD22-positive cell using a midi MACS device and LS columns(Miltenyi) followed by selection of phycoerythrin- and APC-negativecells using a MoFlo cell sorter (DakoCytomation, Fort Collins, USA).CD22-positive IgM-, IgD-, IgA-negative B cells were then incubated withEBV containing supernatant obtained from B95-8 cells in the presence ofCpG 2006 (6, 15) in B cell medium containing RPMI 1640 supplemented with10% fetal calf serum. Cells were seeded in at 50 cells per well in Bcell medium on 30.000 irradiated feeder PBL prepared from voluntarydonors.

After 2 weeks of culture the conditioned medium of memory B cellcultures was screened for the presence of NY-ESO-1-specific antibodiesby ELISA and on NY-ESO-1-positive autologous and non-autologous tissuesections.

ELISA

96 well strip well microplates (Corning, N.Y., USA) were coated with 25μl/well of a 1 μg/ml recombinant NY-ESO-1 protein in PBS overnight at 4°C. Plates were washed with PBS-T and blocked overnight at 4° C. with PBScontaining 5% milk powder (Rapilait, Migros, Switzerland). B cellconditioned medium, patient serum and recombinant antibody preparationswere incubated for 2 h at room temperature. Binding of human antibodiesto NY-ESO-1 was determined using a donkey anti-human IgG-HRP secondaryantibody (Jackson ImmunoResearch Europe Ltd., Cambridgeshire, UK)followed by measurement of the HRP activity using a TMB substratesolution (TMB, Sigma, Buchs, Switzerland).

Epitope Mapping ELISA

20mer peptides spanning the entire NY-ESO-1 protein with 10 aa overlapsshared by each adjacent peptides (Peptides&Elephants, Nuthetal, Germany)were used to coat Maxisorp ELISA plates (Nunc, Rochester, N.Y.). Humanrecombinant antibody Manhattan or patient serum (diluted 1:500 in PBS)was detected using horseradish peroxidase-conjugated Goat anti-humanIgG+IgM (Jackson ImmunoResearch).

Competition ELISA

Saturation experiments identified the half-maximal binding concentrationof human monoclonal antibody Manhattan to NY-ESO-111-30 peptide as1×10⁻⁹ M or 0.15 μg/ml. In competition experiments, increasingconcentrations of NY-ESO-111-30 peptide were mixed with Manhattan at aconcentration of 0.15 μg/ml and the mix was then transferred to ELISAplates coated with NY-ESO-111-30.

Immunohistochemistry

Cylinders of tumor tissues measuring 0.6 mm in diameter were punched outof paraffin embedded NY-ESO-1-positive tumor tissue and healthy controltissue. Pairs formed of a cylinder of tumor tissue and of healthycontrol tissue were placed at each position of a 2×4 grid whosedimensions were compatible with the microtiter format of the B cellculture plates and conventional multi channel pipettes.

Immunohistochemistry was performed on formalin-fixed, paraffin-embeddedtissue. Heat-based antigen retrieval was applied to all slides.Non-specific fluorescence was blocked using polyclonal rabbit anti-humanIgG (Dako, Baar, Switzerland) for 30 min at room temperature followed bya second block in 1% of low fat milk (Rapilait, Migros, Switzerland) for10 min. Primary antibody or B cell conditioned medium was incubatedovernight at 4° C. Binding of human antibodies to NY-ESO-1 was revealedusing Cy 3-conjugated secondary antibodies to human IgG (JacksonImmunoResearch Europe Ltd., Soham, UK). Staining of biotinylatedrecombinant human antibody Manhattan was revealed using Cy3- orHRP-conjugated streptavidin (Sigma, Buchs, Switzerland). As positivecontrol for the presence of NY-ESO-1 antigen a mouse anti-NY-ESO-1monoclonal antibody (Zymed, South San Francisco, USA) was used.

Analysis of immunofluorescence was performed on an inverted fluorescencemicroscope (Leica, Heerbrugg, Switzerland).

Single Cell-RT-PCR

Single cells obtained from a memory B cell culture were deposited intoPCR tubes. cDNA was prepared using primers specific for the constantregions of immunoglobulin G heavy, κ-light and γ-light chains. PCRamplification of immunoglobulin heavy and light chain variable regionswas performed according to standard protocols (7, 16). Immunoglobulinheavy and light chain variable regions were amplified using a seminested PCR approach. 1st round PCR was performed with primers specificfor the IgG constant region and pools of primers specific for conservedframework 1 regions of heavy and light chain Ig variable region families(7). Subsequently, semi-nested PCR with nested primers specific for theIgG constant region and primers specific for framework 1 of heavy andlight chain Ig variable region families that contained restriction siteswere used as described (8). The immunoglobulin heavy and light chain PCRproducts were cloned into vectors containing the constant region ofIgG1, IgKappa or IgLambda.

Antibody Production and Purification

293-T human embryonic kidney cells were cultured in DMEM supplementedwith 10% ultra-low IgG FCS, 1% penicillin-streptomycin and 1% L-glutamin(Invitrogen, Basel, Switzerland). Co-transfection with immunoglobulinheavy and light chain encoding plasmid DNA was performed by the standardcalcium phosphate precipitation method. Thereafter the cells werecultured in serum free D-MEM supplemented with 1% Nutridoma SP (Roche,Rotkreuz, Switzerland). Supernatants were collected after 8 days ofculture and IgG was purified on a protein G column (AmershamBiosciences, Upsala, Sweden) using fast protein liquid chromatography(FPLC) (Amersham Biosciences, Upsala, Sweden). Purified Manhattanantibody was biotinylated following the manufacturers instructions(SIGMA, Buchs, Switzerland).

Immunofluorescence SK-MEL-37 Tumor Cells

SK-MEL-37 cells were grown onto microscope slide, fixed withformaldehyde and permeabilized with 1% Triton X-100 for 10 min at roomtemperature. After blocking with 10% goat serum for 1 h RT cells wereincubated with Manhattan at a concentration of 1 ug/ml or negativecontrol antibody (hu8-18c5 (17) expressed in recombinant fashion with ahuman Fc region) in PBS/1% goat serum/0.2% Triton X-100 overnight at 4°.Bound antibodies were visualized by staining with goat anti-human IgGAlexa Fluor® 546 (1:300, Molecular Probes, Leiden, Netherland) for 1 hRT. Microscopy was performed using a Leica SP 5 microscope.

Example 1 Identification of NY-ESO-1-Specific B Cells from PBL of aMelanoma Patient

A melanoma patient was selected with a serum titer to the taa NY-ESO-1in ELISA and on autologous lymph node sections obtained at biopsy. Postvaccination with recombinant vaccinia virus expressing full lengthNY-ESO-1 a partial clinical response demonstrated by the regression oftwo NY-ESO-1-positive metastases in the liver was observed. 50 mlPeripheral blood was collected from the patient and surface IgM/IgDdouble-negative B cells representing the Ig-switched memory B cells wereisolated and cultured after immortalization using a modified EpsteinBarr virus transformation protocol (6). 100.000 memory B cells wereobtained and were seeded into 96 well microtiter templates at 50 cellsper well. After 3 weeks of culture growing clones were observed in theculture wells and the medium conditioned by the B cell cultures wasassayed for the presence of antibodies specific to NY-ESO-1. As a firstscreening an ELISA using recombinant full length NY-ESO-1 as antigen wasperformed. ELISA signals were rated as positive if they exceeded thebackground signal by a factor of three. This identified 9 ELISA-positivememory B cell culture wells out of the 2000 wells total. An example ofthe signal to noise ratio obtained with the ELISA is depicted in FIG.1A. The ELISA-positive cultures were subsequently assayed inimmunohistochemistry using NY-ESO-1-positive tumor tissue. The setup ofthe tissue screen consisted of 8 pairs of tissue rods ofNY-ESO-1-positive mamma tumors and healthy mamma tissue as controlsmounted on to glass slides. Due to the miniaturization of this assay 15μl of B cell conditioned medium were sufficient to perform the assay.The ability to compare the conditioned medium of several memory B cellcultures and of negative controls on a single slide facilitated theevaluation of the fluorescence staining.

The evaluation of the 9 ELISA-positive B cell cultures in this tissueassay identified one culture that yielded a higher staining intensity ascompared to that of the other 8. This is illustrated in FIG. 1B, whereimmunofluorescence obtained with tissue-reactive culture 12D7 iscompared to immunofluorescence obtained with well 9D1 which was rated asbeing not tissue-reactive.

Since IgG-subclass information on the NY-ESO-1-specific antibody wouldhave been lost in the molecular cloning step it was determined at thisstep using immunohistochemistry with NY-ESO-1-positive tissue sectionsin combination with subclass-specific secondary antibodies anti humanIgG1, IgG2, IgG3 and IgG4. As shown in FIG. 1C, tissue staining forNY-ESO-1 is only observed with a secondary antibody anti IgG1.

Example 2 Molecular Cloning of an NY-ESO-1-Specific Antibody Secreted byCultured Memory B Cells

Previous attempts at the cellular cloning of identified antigen-specificEBV-transformed human memory B cells had not been successful. Therefore,in accordance with the present invention it was embarked on a molecularcloning strategy based on RT-PCR of single sorted cells harvested fromwell 12D7 in order to isolate the antibody clone responsible for theabove described staining pattern. 32 cells were harvested and depositedas single cells directly into PCR tubes.

After cDNA synthesis, the heavy and light chain variable regions ofhuman immunoglobulin were amplified using a nested PCR approach (7).Heavy and kappa light chain sequences with 16 of the 32 sorted cellswere obtained. PCR for lambda light chain variable sequences did notgive a product with any of the cells. Sequence analysis identified 4distinct antibody clones which were numbered according to their relativeabundance. Clone 1 was found in eight of the 16 cells, clone 2 in fourcells and clones 3 and 4 each in two cells.

It was then determined whether one of these four clones when expressedas recombinant antibody yielded a similar NY-ESO-1 staining as observedwith conditioned medium from B cell culture well 12D7. To that end, theheavy and light chain variable sequences were cloned into antibodyexpression vectors that provided the constant regions of the human IgG1heavy chain and of the human kappa light chain (8). The constant regionsof IgG1 were used since the NY-ESO-1-specific antibody identified inconditioned medium of well 12D7 was determined to be of this subclass(FIG. 1 C).

Functional analysis of the four clones was performed by re-screening therecombinant antibodies in ELISA and on NY-ESO-1-positive tissuesections. To that end, heavy chain and corresponding light chainexpression vectors of the four clones were transfected into 293 HEKcells and the supernatant fluid of the transfected cells was testeddirectly in ELISA and immunohistochemistry. All four supernatant fluidsproduced functional IgG1 as tested in anti-human-IgG-ELISA. While clones1-3 did not show any binding to NY-ESO-1 in ELISA clone number 4 waspositive up to the last dilution tested ( 1/100) (FIG. 2 A). This clonealso showed a specific staining in immunohistochemistry usingNY-ESO-1-positive tissue sections (FIG. 2 B).

This was taken as confirmation that the sequence of the immunoglobulinvariable regions of the original NY-ESO-1-specific antibody as itoccurred in the patient had been retrieved. For the sake of simplicityclone 12D7 No. 4 was named “Manhattan” and used for furthercharacterization using protein G purified material obtained fromtransiently transfected HEK cells.

Example 3 NY-ESO-1 Specific Human Monoclonal Antibody Manhattan Binds topeptide NY-ESO-1₁₁₋₃₀ with a KD of 10⁻¹⁰

To identify the epitope recognized by Manhattan on NY-ESO-1 ELISA wasperformed using overlapping peptides spanning the complete NY-ESO-1protein. As shown in FIG. 3A, Manhattan binds to a peptide representingthe amino acids 11 to 30 from the NY-ESO-1 protein but not to the twoadjacent peptides that span amino acids 1-20 or 21-40. This suggeststhat the epitope recognized by Manhattan lies at the junction of thesetwo peptides around amino acid 20 of NY-ESO-1. This epitope, amongothers was also recognized by antibodies contained in serum of patientC1 (FIG. 3B).

The avidity of Manhattan was determined by competition ELISA usingincreasing concentrations of soluble NY-ESO-1₁₁₋₃₀ peptide to competefor the plate bound peptide. As depicted in FIG. 3C, the antigen-bindingequilibrium dissociation constant (KD) of the interaction of Manhattanwith its cognate peptide was in the lower nanomolar range.

As a final assay used in the characterization of human monoclonalantibody Manhattan immunofluorescence analysis on the NY-ESO-1-positivecell line SK-MEL-37 was performed (9). Staining of this cell line withManhattan resulted in a nuclear signal that co-localized with stainingobtained with the nuclear marker Hoechst.

CONCLUSION

The above experiments provide a general method for the identificationand molecular cloning of antibodies directly from peripheral bloodlymphocytes (PBLs) of human subjects. The method of the presentinvention could be proven by isolating a human monoclonal antibody tothe tumor-associated antigen NY-ESO-1 from a melanoma patient. Startingwith the screening of antibodies secreted by cultures of short termimmortalized human memory B cells cultures that were positive in ELISAand in immunohistochemistry on NY-ESO-1 positive tissue were identified.This primary screen was followed by a molecular cloning step the purposeof which was to identify and isolate the single clone of B cells thatsecreted the antibody detected in the primary screening. The presence ofonly 4 different clones in well 12D7 as revealed by sequence analysisafter single-cell RT-PCR, suggests, that of the initially 50 cells thatwere seeded only few had been immortalized and survived.

A subsequent secondary screen of the recombinant candidate antibodiesresulted in the identification of a single monoclonal antibody with anidentical staining pattern as the original antibody that was produced bythe cultured memory B cells derived from patient PBL. Thus, an antibodyas it occurred originally in the patient could successfully beretrieved. This antibody, coined “Manhattan” recognizes a N-terminalepitope around amino acid position 20 which is shared between NY-ESO-1and the taa LAGE-1 (9). This epitope is also recognized by serum ofpatient C1 supporting the notion of Manhattan as being a genuine copy ofan antibody that occurred in the patient.

As of to date Manhattan is the first human monoclonal antibody toNY-ESO-1 it may also be the first patient-derived affinity maturedantibody to a tumor antigen and taa, respectively. This novel method ofthe present invention bypasses some of the difficulties inherent toEBV-transformation of B cells such as genetic instability and poorcloning efficiency (6, 10). While the isolation of human monoclonalantibodies from EBV-immortalized memory B cells had been successfullyperformed in a previous study (6), it is noteworthy to mention thatprevious attempts tried prior to the above described method of thepresent invention at the isolation of NY-ESO-1 specific antibodies fromthe same patient using EBV-transformation and cellular cloningtechniques failed despite a considerable number of memory B cellcultures identified in the cellular screening.

A second object of the present invention was the isolation of anantibody to the tumor-associated antigen NY-ESO-1 withtissue-reactivity. This was motivated by the observation, that serum ofthe patient contained antibodies that reacted with NY-ESO-1-positiveautologous tissue taken at biopsy. To that end the micro-arraytechnology was adapted for the screening of memory B cell cultures. Thishad several advantages as compared to classical methods ofimmunohistochemistry. First, the availability on one single slide ofseveral replica positions allowing to assay and to compare severalsamples. Second, the possibility to place positive tissue adjacent tonegative tissue greatly improves assay sensitivity, a feature which wascrucial since incubation with conditioned medium of memory B cellcultures often resulted in very weak staining. Third, thisminiaturization of the assay format needs much less of conditionedmedium which also is a decisive factor since the culture volume ofmemory B cell cultures was generally less than 200 μl.

The observation that serum of the patient and human monoclonal antibodyManhattan recognized fixed tissue sections may be irrelevant for thesituation in vivo at least with regard to a direct therapeutic role viathe induction of antibody induced immune effector mechanisms acting on acellular level to clear NY-ESO-1-positive cells. NY-ESO-1 has beendescribed as an intracellular antigen (11) and was shown in this studyto be localized in the nucleus, at least in the cell line SK-Me-37. Inthis context, surface staining on live SK-Me-37 using biotinylatedManhattan had been negative.

The isolation of Manhattan constitutes a major step towards theevaluation of the therapeutic significance of patient-derivedtumor-specific antibodies. There are several scenarios conceivableaccording to which such an antibody could mediate therapeutic effects.First, it could serve as an adjuvant for future vaccine protocols.Immune complexes formed upon co-administration of Manhattan withNY-ESO-1 could result in an increased induction of cellular immuneresponses (12).

A second possibility addresses the pathophysiological role of this classof antibodies in tumor patients. NY-ESO-1 frequently induces humoralresponses which correlate with a bad prognosis for the patient (13).While this could be a mere correlation due to increased abundance ofantigen as the tumor grows, a tolerogenic role of this B cell responsecould also be hypothesized. According to this scenario, free antigenreleased by necrotic or apoptotic tumor cells would induce a strong Bcell response, the B cells then would present antigen as a result ofFc-receptor-mediated uptake of immune complexes (14). As B cells may bepoor APC, this presentation could result in the induction of toleranceof NY-ESO-1-reactive T cells and thus prevent tumor rejection. Theadministration, in an early phase of tumor progression, of recombinantManhattan F(ab)s could disrupt the uptake of antigen by B cells becauseF(ab) are not bound by Fc-receptors but would still capture antigen.This in turn, could prevent the tolerance induction in NY-ESO-1-specificT cells.

REFERENCES

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1. A method of isolating an antibody specific for a tumor antigen ortumor-associated antigen comprising: (a) subjecting a sample obtainedfrom a patient bearing a tumor antigen or tumor-associated antigenpositive tumor, wherein said patient shows a at least partial clinicalresponse or is symptom-free, to a specimen of tumor cells or tissue ofpredetermined clinical characteristics; and (b) identifying andisolating an antibody which binds to said specimen but not to cells ortissues of a healthy subject.
 2. The method of claim 1, wherein saidsample comprises or is derived from peripheral blood.
 3. The method ofclaim 1, wherein said sample comprises or is derived from B-cells ormemory B-cells.
 4. The method of claim 1, wherein each of said patientand said healthy subject is a human.
 5. (canceled)
 6. The method ofclaim 1, wherein said specimen is derived from a tumor patient or ananimal model for a human tumor.
 7. The method of claim 1, wherein saidtumor-associated antigen is selected from the group consisting of SCP-1,SSX-4, HOM-TES-85/CT-8, GAGE, SSX-1, SSX-2, NY-BR-1, LAGE-1 andNY-ESO-1.
 8. The method of claim 1, wherein said specimen comprisesautologous tumor tissue or cells of said patient.
 9. The method of claim1, wherein said patient is a melanoma patient and said specimencomprises autologous sections derived from a lymph node metastasis. 10.The method of claim 1, further comprising the steps of: (i) purifyingB-cells or memory B-cells from a sample which has been identified tocontain binding molecules, i.e. antibodies which bind to said specimenbut not to corresponding cells or tissue of a healthy subject; (ii)obtaining the immunoglobulin gene repertoire for said antibodies fromsaid B-cells or memory B-cells; and (iii) using said repertoire toexpress said antibodies.
 11. The method of claim 10, wherein step (ii)comprises the steps of: (iv) obtaining mRNA from said B-cells or memoryB-cells; (v) obtaining cDNA from the mRNA of step (iv); and (vi) using aprimer extension reaction to amplify from said cDNA the fragmentscorresponding to the heavy chains (HC) and the kappa light chains (LC)of said antibodies.
 12. The method of claim 10, wherein a single-cellRT-PCR is employed for obtaining the immunoglobulin gene repertoire forsaid antibody.
 13. (canceled)
 14. A tumor-specific antibody obtainableby the method of claim 1 or a binding fragment thereof.
 15. The antibodyor binding fragment of claim 14, which is capable of selectivelyrecognizing a tumor antigen or tumor-associated antigen.
 16. Theantibody or binding fragment of claim 15, wherein said antigen isselected from cancer testis (CT) antigens.
 17. The antibody or bindingfragment of claim 15, wherein said antigen is selected from the groupconsisting of SCP-1, SSX-4, HOM-TES-85/CT-8, GAGE, SSX-1, SSX-2,NY-BR-1, LAGE-1 and NY-ESO-1.
 18. The antibody or binding fragment ofclaim 15, wherein said antigen is tumor-associated antigen NY-ESO-1. 19.The antibody or binding fragment of claim 14, selected from the groupconsisting of a single chain Fv fragment (scFv), a F(ab′) fragment, aF(ab) fragment, and an F(ab′)₂ fragment.
 20. A tumor-specific antigenwhich is recognized by the antibody or binding fragment of claim
 14. 21.A polynucleotide encoding at least the variable region of animmunoglobulin chain of the antibody or binding fragment of claim 14.22. A vector comprising the polynucleotide of claim
 21. 23. A host cellcomprising a polynucleotide of claim
 21. 24. A method for preparing anantibody or a binding fragment or immunoglobulin chain(s) thereof, saidmethod comprising: (a) culturing the cell of claim 23; and (b) isolatingsaid antibody or binding fragment or immunoglobulin chain(s) thereoffrom the culture.
 25. (canceled)
 26. The antibody or binding fragment ofclaim 14, which is detectably labeled.
 27. The antibody or bindingfragment of claim 26, wherein the detectable label is selected from thegroup consisting of an enzyme, a radioisotope, a fluorophore and a heavymetal.
 28. The antibody or binding fragment of any one of claim 14,which is attached to a drug.
 29. A composition comprising the antibodyor binding fragment of claim
 14. 30. The composition of claim 29 furthercomprising a pharmaceutically acceptable carrier.
 31. The composition ofclaim 30 further comprising an additional agent useful for treatingtumors.
 32. A diagnostic composition comprising the antibody or bindingfragment of claim
 14. 33-34. (canceled)
 35. A method of treating orpreventing the progression of a tumor in a subject, for ameliorating thesymptoms associated with a tumor; for diagnosing or screening a subjectfor the presence of a tumor or for determining a subject's risk fordeveloping a tumor, which method comprises administering to said subjectan effective amount of the antibody or binding fragment of claim
 14. 36.(canceled)
 37. A method of diagnosing and/or treating a disorder relatedto a tumor comprising administering to a subject a therapeuticallyeffective amount of a tumor antigen binding molecule comprising at leastone CDR of the antibody or binding fragment of claim 14 or acorresponding anti-idiotypic antibody. 38-40. (canceled)